Abstract
SummaryMouse embryonic stem cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1 phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here, we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and that is required for the elongated G1 phase characteristic of ground state ESCs. RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that P53 directly regulates the expression of the retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveal a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation.
Highlights
Mouse embryonic stem cells (ESCs) are pluripotent and self-renewing cells derived from the inner cell mass of the mouse blastocyst
A major pathway employed by P53 to prevent DNA damage is by halting G1-phase progression and S-phase entry via promoting Cdkn1a expression, which results in the inhibition of the CYCLIN/CDK complexes
The elevated expression of P21 and elongated G1 phase in 2i ESCs (Ter Huurne et al, 2017) led us to hypothesize that P53 is active in 2i ESCs, but not in serum ESCs, and contributes to cell cycle regulation in the pluripotent ground state
Summary
Mouse embryonic stem cells (ESCs) are pluripotent and self-renewing cells derived from the inner cell mass of the mouse blastocyst. Mouse ESCs cultured in chemically defined 2i medium (N2B27 with PD0325901, CHIR99021, and LIF, hereafter called 2i ESCs) (Ying et al, 2008) were shown to have an unrestricted developmental potential and are hypothesized to represent the ground state of pluripotency (Habibi et al, 2013; Marks et al, 2012). We have previously shown that the short G1 phase is characteristic of serum ESCs and is the result of ERK signaling The latter pathway is inhibited in ground state pluripotent ESCs cultured in 2i, resulting in an elongated G1 phase (Ter Huurne et al, 2017). P21 and P27 prevent CDK-mediated phosphorylation and inactivation of the pocket proteins, and thereby activate the G1 checkpoint
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